Butanol is an important industrial chemical with a variety of applications, where its potential as a fuel or fuel additive is particularly significant. Butanol is favored as a fuel or fuel additive because it yields only CO2 and little or no SOx or NOx when burned in the standard internal combustion engine. Although butanol is a four-carbon alcohol, it has an energy content similar to that of gasoline and can be blended with any fossil fuel. Additionally, butanol is less corrosive than ethanol, the most preferred fuel additive to date.
Butanol also has the potential of impacting hydrogen distribution problems in the fuel cell industry. Fuel cells today are plagued by safety concerns associated with hydrogen transport and distribution. Butanol, however, can be easily reformed for its hydrogen content and can be distributed through existing gas stations in the purity required for either fuel cells or vehicles. Butanol is also useful as a feedstock chemical in the plastics industry and as a food grade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase in the future.
Methods for the chemical synthesis of isobutanol are known, such as oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, Wiley-VCH Verlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719 (2003)) and Guerbet condensation of methanol with n-propanol (Carlini et al., J. Molec. Catal. A. Chem., 220:215-220 (2004)). These processes use starting materials derived from petrochemicals, are generally expensive and not environmentally friendly. The production of isobutanol from plant-derived raw materials could minimize the use of fossil fuels.
Isobutanol is produced biologically in minute quantities as a by-product of yeast fermentation. It is a minor component of “fusel oil” that forms as a result of the incomplete metabolism of amino acids by yeast. Isobutanol is specifically produced from catabolism of L-valine. After the amine group of L-valine is harvested as a nitrogen source, the resulting α-keto acid is decarboxylated and reduced to isobutanol by enzymes of the so-called Ehrlich pathway (Dickinson et al., J. Biol. Chem. 273:25752-25756, 1998) (“Dickinson”). Addition of exogenous L-valine to the fermentation medium increases the yield of isobutanol, as described by Dickinson, wherein it is reported that a yield of isobutanol of 3 g/L is obtained by providing L-valine at a concentration of 20 g/L in the fermentation broth. However, the use of valine as a feedstock would be cost prohibitive for industrial scale isobutanol production.
Microorganisms expressing engineered biosynthetic pathways for producing butanol, including isobutanol, directly from sugars have been described previously in, e.g., U.S. Pat. Nos. 7,851,188 and 7,993,889. Such butanologens may further include disruption of certain genes involved in the formation of by-products during fermentation in order to maximize the yield of butanol isomers. The genes involved in the by-product formation include the genes necessary for ethanol formation (see U.S. Patent Appl. Pub. No. 20090305363) and isobutyric acid formation. (see PCT Patent Appl. Pub. No. WO2012/129555). Microorganisms in which genes necessary for ethanol formation (e.g., PDC gene) are disrupted require an exogeneous C2 supplement for proper growth. This requirement for a C2 supplement is usually met by adding small amounts of ethanol to the culture medium. For example, U.S. Patent Application Publication No. 20090305363, incorporated herein by reference, describes PDC knockout yeast strains that were unable to grow in a medium containing 2% glucose as carbon source, but were found to grow very well in a medium containing glucose supplemented with a small amount of ethanol.
Under some circumstances, butanologens with disruptions in genes necessary for isobutyric acid production (e.g., ALD6) in addition to PDC gene disruptions may have altered ability to grow and produce butanol, even when ethanol is used as a C2 supplement. Although approaches to such challenges have been described in the art, for example by engineering the strain for reduced C2 dependence (see, for example, US App. Pub. No. 20120156735), alternative or supplemental methods to replace or supplement such strategies would represent an advance in the art.